The mammalian genome contains about 3 x 10 methylated cytosine residues. Genomic methylation patterns are essential for genome stability, for allele-specific expression of imprinted genes, for X chromosome inactivation in females, and for transcriptional repression of the transposons and their remnants that make up more than 45% of the mammalian genome. Global genome demethylation is lethal to differentiated cells, and focal de novo methylation or demethylation is involved in a variety of developmental abnormalities and carcinogenesis in humans. Methylation patterns are established and maintained by DNA (cytosine-5)-methyltransferases. These enzymes are highly conserved and typically display 10 conserved motifs whose catalytic functions are known from crystallographic studies. Mammals and many other taxa (including some whose genomes are not known to be methylated) express a protein (DNA methyltransferase-2 or Dnmt2) that must be a DNA methyltransferase by every detail of sequence and structure but which does not display +transmethylase activity in vitro, nor do embryonic stem cells that tack Dnmt2 display detectable abnormalities of genomic methylation patterns. Unlike known DNA methyltransferases, Dnmt2 cleaves and binds to DNA in an irreversible covalent complex. This may be mediated by a motif related to the active site of type 1A topoisomerases which is not found in other methyltransferase homologues. The Dnmt2 gene has very recently been shown to be a recurrent site of integration of a Moloney-related murine leukemia virus in mouse leukemia and lymphoma, and the chromosomal region that contains the human DNMT2 gene (10p12-10p13) is frequently rearranged in human leukemia and lymphoma. DNMT2 has also been reported to be downregulated in certain carcinomas. We will perform biochemical and genetic experiments that will determine the biological functions of Dnmt2 in normal cells, and will determine whether the Dnmt2 gene is altered in neoplastic diseases of humans.